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Isolation and Identification of Pandoraea Spp. From Tabatabaei et al. Italian Journal of Pediatrics (2019) 45:118 https://doi.org/10.1186/s13052-019-0687-x RESEARCH Open Access Isolation and identification of Pandoraea spp. From bronchoalveolar lavage of cystic fibrosis patients in Iran Mohammad Tabatabaei1*, Mahdi Dastbarsar1 and Mohammad Ashkan Moslehi2 Abstract Background: Pandoraea species are gram negative, motile, non-spore forming, rod shaped and oxidase positive, obligate aerobes bacteria, and have one polar flagellum. Most of Pandoraea species are associated with lung infections in cystic fibrosis patients. Cystic fibrosis is the most prevalent autosomal recessive hereditary disease in the world that affects various organs of the body. The main important cause of death in these patients is lung involvement. This study was conducted to isolate and identify Pandoraea bacterium from bronchoalveolar lavage and sputum samples of cystic fibrosis patients in Shiraz, Iran. Methods: In this research 31 samples of bronchoalveolar lavage and sputum were examined by culture and PCR method. Then confirmed isolates were evaluated for susceptibility to different antibiotics and ability to produce biofilm. Results: The results of this study after cultivation, purification and DNA extraction led to the isolation of 4 Pandoraea bacterium by PCR using specific primers. Antibiotic susceptibility test were indicated all isolates were resistant to gentamicin, amikacin and imipenem and susceptible to ciprofloxacin, trimethoprim-sulfumethoxazole, piperacillin and tetracycline. Ability to create biofilm was indicated by some of Pandoraea isolates. According to findings of this study, ability to synthesis biofilm by Pandoraea isolates and resistance to some antibiotics are very important. Conclusions: Our study notes the role of P. pnomenusa as an emerging pathogen that can cause chronic lung colonization in CF patients. Identification tools need to be accurate and must be based on molecular techniques. Also our findings should raise awareness about antibiotic resistance in cystic fibrosis patients in Iran and ability of including bacterial agents to produce biofilm is an alarm for public health. Thus clinicians should exercise caution about finding of clinical relevance of this pathogen to the infection and prescribing antibiotics, especially in cases of children infections. Keywords: Pandoraea spp., Cystic fibrosis, Bronchoalveolar lavage, PCR, Iran Introduction faecigallinarum, P. vervacti, P. thiooxidance and four un- The genus Pandoraea is composed of aerobic, non-spore- named genomospecies. The majority of isolates have been forming, non-nitrate reducing, non-lactose fermenting, and isolated from respiratory samples from patients with cystic gram negative rods shaped bacteria, with polar flagella. fibrosis (CF) or other underlying chronic lung disease but They have been grown at 30–37 °C in 0.5 and 1.5% NaCl can also be isolated from other clinical samples (including or on Drigalski agar, assimilate D-gluconate, L-malate, and blood) and from the environmental samples such as soil, phenylacetate, and have catalase, acid and alkaline phos- food, sea, and drinking water [1–5]. Its closest phylogenetic phatase and leucine arylamidase activity [1]. Different spe- relative is the genus Burkholderia and, like members of the cies of Pandoraea have been described: P. apista, P. genus Burkholderia,thePandoraea spp. are emerging im- norimbergensis, P. pnomenusa, P. pulmonicola, P. spu- portant respiratory pathogens, particularly in people with torum, P. fibrosis, P. terrae, P. oxalativorance, P. cystic fibrosis (CF). Species in this genus are often misiden- tified as Burkholderia cepacia complex (Bcc) or Ralstonia * Correspondence: [email protected] species owing to overlapping biochemical profiles without 1Faculty of Veterinary Medicine, Shiraz University, Shiraz, Iran differences that reliably distinguish between species [6]. It Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tabatabaei et al. Italian Journal of Pediatrics (2019) 45:118 Page 2 of 8 is because of these limitations that reliable identification re- Whether Pandoraea spp. is truly pathogenic is not yet quires 16S ribosomal DNA sequence analysis. fully understood, there is evidence that infection with Pan- Cystic fibrosis is caused by mutations in the CFTR (cys- doraea spp. invokes a host inflammatory response. A num- tic fibrosis transmembrane conductance regulator) gene ber of virulence factors have been associated with clinical [7]. The commonest mutation is the deletion of phenyl- virulence of Pandoraea spp. and Bcc, including stimulation alanine at codon 508 (phe508del). This occurs in about of pro-inflammatory cytokine secretion known to cause 70% of patients with cystic fibrosis. The primary function lung tissue damage (IL-6 and IL-8), biofilm formation and, of the CFTR protein is as an ion channel that regulates li- in the case of Bcc, the ability to invade lung epithelial cells, quid volume on epithelial surfaces through chloride secre- which may contribute to the persistence of the strains in tion and inhibition of sodium absorption. The commonly the CF lung [16–20]. Indeed, biofilm formation by Pandor- accepted explanation for airway disease in cystic fibrosis is aea spp. and Bcc has been specifically associated with in- the “low volume” hypothesis. A reduced volume of airway creased resistance to antibiotics and maintenance of the surface liquid causes failure of mucociliary clearance, the bacteria in the lung [21–24]. Here, we report the results of lungs’ innate defense mechanism [8]. The mucociliary dys- isolation and identification of Pandoraea spp. and their function means that a patient with CF cannot effectively ability to form biofilms in vitro, in order to gain insight into clear inhaled bacteria. In addition, there is an excessive in- their virulence potential in CF lung infections. flammatory response to pathogens. For a given bacterial In the clinical microbiology laboratory, identification to load, a person with CF will have up to 10 times more in- the species level and differentiation of Pandoraea species flammation than a person with a lower respiratory tract from organisms belonging to the Bcc, R. pickettii,orR. infection but without the disease. The reasons for the ex- paucula may be problematic [1, 6, 25]. To aid in the iden- cessive inflammatory response to pathogens are not fully tification of these organisms, we used PCR-based identifi- understood. The abnormal composition and secretion of cation strategies based on the 16S rRNA gene (rDNA). mucus may also be important. At birth, the airway is unin- fected and probably uninflamed [9], but the end result of Materials and method the abnormalities described above is irreversible airway Sample collection damage with bronchiectasis and respiratory failure in most From November 2017 to August 2018, following the sed- patients. Ion and water abnormalities may also cause dis- ation of CF patients in Namazi hospital (Shiraz, Iran), 31 ease in other epithelia-lined organs. samples of bronchoalveolar lavage and sputum were col- The main source of morbidity and mortality in CF pa- lected. Then the samples were transferred to the bacteri- tients, is the decline in the pulmonary function subsequent ology laboratory, faculty of veterinary medicine of Shiraz to pathogenic colonization with non-fermenting Gram University, in cold condition. The study was approved by negative bacteria (NFGNB) that they encounter throughout the Ethics Committee of Shiraz University of Medical their lives.CF patients are particularly susceptible to infec- Sciences. tions caused by specific bacterial pathogens such as Pseudo- monas aeruginosa, Staphylococcus aureus and Haemophilus Culture and isolation influenzae [10]. To obtain strains and to compare culture and PCR re- Although Pandoraea species have also been isolated sults, cultures were performed initially as enrichment from sputum samples of CF patients, there is still very cultures. Upon arrival, bronchoalveolar lavage and spu- little known about their mechanisms of pathogenicity or tum were cultured in BHI broth and incubated aerobic- their roles in CF lung disease [11]. In addition, Pandor- ally at 37 °C for 24 h. Initial enriched cultures were aea isolates have been recovered from both CF and non- transferred to the enrichment culture plates by serial CF patients from a variety of clinical samples including streaking the cultures on sheep blood agar and incu- blood, sputum, urine, the upper airways and lung tissue bated aerobically at 37 °C for 24–48 h. [12]. The recovery of Pandoraea isolates from the blood Then, a loop full of the identified colonies were streaked of patients indicates that this organism is capable of in- on a new blood agar and incubated aerobically at 37 °C for vading tissue [13, 14]. Antibiotic therapy for treatment 24 h. Next, from culture positive plates, typical colonies
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